I thought I had figured this one out. But apparently not.
I need to convert a BAM file of paired-end alignments to two FASTQ files of paired reads to realign them, with a twist: I only want reads that fall within a defined region.
Currently I am using the following command (the region is an example; it also fails on different regions; at any rate, this is on hs37d5 but presumably the issue would exist for other references too):
samtools view -b -u -@ 48 alignments.bam 21:31,831,502-31,896,094 \ | samtools collate -n 128 -u -O -@ 48 - /tmp/bam-to-fastq- \ | samtools fastq -F 0x900 -@ 48 \ -0 /dev/null -1 reads_R1.fastq.gz -2 reads_R2.fastq.gz -
(Apart from the region filtering, this command is essentially the same that word of god tells us to use.)
The resulting files are not properly paired:
⟩⟩⟩ diff <(gunzip -c reads_R1.fastq.gz | sed -n -e 1~4p -e 10q) \ 1 ⟩ <(gunzip -c reads_R2.fastq.gz | sed -n -e 1~4p -e 10q) 1,2d0 < @A00346:17:H52H7DSXX:3:2142:32316:24518 < @A00346:17:H52H7DSXX:3:1153:6705:26412 3a2,3 > @A00346:17:H52H7DSXX:3:1173:9118:3129 > @A00346:17:H52H7DSXX:3:1109:29414:11757
As far as I can see from performing a spot check, these are all reads which are marked as paired in the BAM file, but for which only one mate falls within the filtered region.
bwa mem on the files gives me the error:
[mem_sam_pe] paired reads have different names: […]
How can I ensure that only properly paired reads are included in the FASTQ files?